Baciphelacin: a new eukaryotic translation inhibitor

Biochimie, 69 (1987) 797- 802 © Soci6t~ de Chimie biologique/Elsevier, Paris

797

Baciphelacin: a new eukaryotic translation inhibitor* Luis CARRASCO

Departamento de Microbiologia; Centro de Biologia Molecular, Universidad Autdnoma, Canto Blanco, 28049 Madrid, Spain (Received 8-1-1987, accepted after revision 5-5-1987)

Summary - Baciphelacin an antibiotic produced by Bacillus thiaminolyticus was a potent inhibitor of protein synthesis in HeLa cells and other mammalian cell lines. It had no effect on DNA or RNA synthesis. Concentrations of baciphelacin around 10 - 7 M inhibited protein synthesis by 50°70 in intact cells. The antibiotic had no effect on protein synthesis in Saccharomyces cerevisiae or Escherichia coli, but inhibited the protozoan Trypanosoma brucei. In vitro protein synthesis in a rabbit reticulocyte cell-free system was blocked by baciphelacin. However, translation of globin mRNA in a wheat cell-free system was not affected by this antibiotic. Baciphelacin had no activity against a number of cell-free systems used to measure different steps of translation, including binding of substrates to the ribosome, peptide bond formation and polyphenylalanine synthesis. Therefore, it is assumed that it affects the initiation of translation or the charging of tRNA. Finally, the inhibition of protein synthesis by compounds structurally related to baciphelacin was tested and their effects compared to baciphelacin. antibiotics / protein synthesis / baciphelacin

R~sum~ - La baciph~lacine: un nouvel inhibiteur de la traduction chez les eucaryotes. La baciph61acine, antibiotique produit par Bacillus thiaminolyticus, est un puissant inhibiteur de la synthb.se prot~ique chez les cellules HeLa. Ce compos6 n'a aucun effet sur la synth~se de I'ADN ou de I'ARN. La traduction est dgalement bloqude par ce composd chez d'autres ligndes ceilulaires de mammif~res. Des concentrations de baciphdlacine d'environ 1O- 7 M inhibent la synth~se protdique de 50% chez les cellules intactes. Cet antibiotique n'a aucun effet sur la synthbse des protdines chez Saccharomyces cerevisiae, ni chez Escherichia coli, mais inhibe le protozoaire Trypanosoma brucei. In vitro, la synth~se protdique avec un syst~me acellulaire de r6ticulocytes de lapin est bloqu6e par la baciphdlccine. Ndanmoins, la traduction d ' A R N messager de globine dans un syst~me acellulaire de bl~ n'est pas affectde par cet antibiotique. La baciphdlacine n'a montr6 aucune activit~ contre un certain nombre de syst~mes acellulaires utilis#s pour mesurer diff~rentes ~tapes de la traduction, et comprenant la fixation de substrats aux ribosomes, la formation de liaisons peptidiques et la synthbse de la polyphdnylalanine. Enfin, nous avons test~ l'inhibition de la synth~se prot~ique par des composds structurellement apparentds ?t la baciphdlacine et leurs effets ont dt~ compares ?t ceux de la baciph~lacine. antibiotiques I synth~se protdique I

baciphdlacine

* This article is dedicated to the memory of my former mentor David Vdzquez.

798

L. Carrasco

Introduction Translation inhibitors have been useful in elucidating the complex process of protein synthesis [1]. Such compounds have been classified into three groups: those that inhibit prokaryotic systems, those that block eukaryotic protein synthesis and those which are active on both processes [1]. Exceptions to this rule are rare. However, compounds that block some euk,'u'yotic systems, but not others, have been described [1]. Tenuazonic acid constitutes a clear example of an antibiotic that selectively binds to mammalian, but not yeast ribosomes and specifically blocks peptide bond formation [2, 3]. During a search for natural compounds active against viral replication, we found a number of them to be very active against mammalian cells [4]. Baciphelacin, an antibiotic produced by Bacillus thiaminolyticus [5], was one such compound [4]. The action of baciphelacin on translation and the effects of a number of other structurally related agents are described.

Cell-free systems for the analysis of protein synthesis Rabbits were made anemic by daily injection of phenylhydrazine and lysed reticulocytes were prepared [8]. Endogenous protein synthesis by the S-30 reticulocyte lysate translation of globin mRNA by wheat germ cellfree systems have been described in detail [9, 10]. Other conditions and methods for analyzing polyphenylalanine synthesis, the non-enzymatic binding of Phe-tRNA and N-Ac-Phe-tRNA to ribosomes and the formation of peptidyl-[3H]puromicin from polysomes, were previously reported [11]. Source of antibiotics We are indebted to several scientists and laboratories for generous gifts of the following compounds: baciphelacin (Takeda Chemical Ind., Osaka, Japan); cladosporin (Dr. P.M. Scott, Sir Frederick Barting Research Center, Canada); frenolicin (Dr. G.A. Ellestard, American Cyanamid Company, USA); 4-acetyl-6,8-dihydroxy5-methyl isocoumarin and 4-acetyl-6,8-dihydroxy5-methyl-3,4-dihydroisocoumarin (Dr. W.B. Turner, Imperial Chemical Industries, U.K.); chlorflavonin and terphenyllin (Dr. L.C. Vining, Dalhousie University, Canada and Dr. M. Richards, Beecham Research Laboratories, U.K.).

Materials and methods Cells HeLa cells, L cells and 3T3 cells were grown in Dulbecco's modified Eagle's medium (E4D) (GIBCO) supplemented with 10070 newborn calf serum (GIBCO). TrvDanosoma hruepi Rarrhar,~myces cerevisiae o,~a Escherichia coil were grown as previously described [6, 7]. Measurement of macromolecular synthesis in cultured cells Cells were grown in 24-well Linbro dishes in 0.5 ml of E4D medium plus 10070calf serum. The antibiotic was added as indicated in each experiment. At the indicated time, the medium was replaced by 0.5 ml of methioninefree medium, supplemented with 1o7/0calf serum. Protein synthesis was estimated by the addition of 2 ~Ci [35S]methionine (800 Ci/mmol; 5.4 mCi/ml). After incubation, the medium was removed and the cell monolayer treated with 0.5 ml of 5°70trichloroacetic acid, washed twice with ethanol and dried under an infrared lamp. The precipitated cell monolayer was then dissolved in 0.2 ml of 0.1 N NaOH, containing 1070SDS. 0.1 ml was withdrawn and counted in a liquid scintillation spectrometer. DNA synthesis was measured by estimating the incorporation of [3H]thymidine (48 Ci/mmol; 1 mCi/ml) and RNA synthesis by the incorporation of [3H]uridine (40 Ci/mmol; 1 mCi/ml), into trichloroacetic acidprecipitable material. The radioactivity incorporated was estimated as described above.

Results E f f e c t s o f baciphelacin on intact eukaryotic The structural formulae of baclphelacin and structurally related compounds are shown in Fig. 1. Fig. 2 shows that baciphelacin has a negligible effect on D N A and R N A synthesis in H e L a cells at concentrations up to 100/zg/ml (2.4 x 10 -4 M), whereas translation was rapidly and efficiently blocked by 2 / z g / m l (5 x 10 -6 M). These results suggest that the primary action of baciphelacin on cellular processes is at the level of the translation machinery. The inhibitory effect of baciphelacin on translation in other cell types, such as normal mouse 3T3 cells and transformed L cells, is shown in Fig. 3.50°70 inhibition was attained at a concentration around 10 -7 M (0.042 ~ g / m l ) baciphelacin. This concentration was similar to that found for HeLa cells (Fig. 3). By contrast, baciphelacin had no effect on translation in S. cerevisiae (Fig. 3), even at 200-fold higher concentrations and was inactive against E. coil translation (Fig. 3). On the other hand, this antibiotic blocked protein synthesis in the protozoan T. brucei.

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To examine if baciphelacin blocked protein synthesis in cell-free systems, we assayed the effect of the antibiotic on the reticulocyte S-30 system. Baciphelacin totally inhibited the synthesis of proteins (mostly globin) in this system (Fig. 4). By contrast, the translation of globin mRNA by a wheat germ cell-free system was not affected. These results indicate that the target of baciphelacin is the translation machinery and suggest that a component of this machinery, different from mRNA, is the target of this antibiotic. As regards the translation step inhibited by baciphelacin, we have evidence that this compound has no effect on the following reactions catalyzed by mammalian systems: poly(U)-directed polyphenylalanine synthesis, non-enzymatic binding of [3H]Phe-tRNA to ribosomes dependent upon poly(U) and [3Hlpuromycin reaction with the growing peptide chains on polysomes (Table I). Based on these results, it is suggested that the step blocked by baciphelacin is located at the initiation of translation, or on the charging of tRNA with amino acids catalyzed by the amino-acyl-tRNA synthetases.

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Fig. 2. Effects of baciphelacin on macromolecular synthesis. DNA, RNA and protein syntheses were measured in HeLa cells as indicated under Materials and Methods. Cells grown as monolayers were incubated with the concentration of baciphelacin indicated in the figure for the times shown.

L. Carrasco

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A number of compounds structurally related to baciphelacin exist in nature. To obtain insight into the structure-activity relationships of these agents, the effect of various compounds related to bacipb-~',cin on protein synthesis was analyzed. Fig. 5 shows that cladosporin, an anti-fungal compound produced by Cladosporium cladosporioides [12], was inactive on protein synthesis in HeLa, or yeast cells. On the other hand, 4-acetyl-6,8-dihydroxy-5-methyl-3,4-dihydroisocoumarin (ADDC), a metabolite of Aspergillus viridinutans [13], actively blocked translation in HeLa cells. Interestingly enough, it also had selectivity against mammalian cells with no effect on yeast. By contrast, a compound related to ADDC, but possessing a double bond between carbons 3 and 4 (see Fig. 1) is devoid of activity (Fig. 5). Chlorflavonin, (a compound related to quercetin) produced by Aspergillus fumigatus [14], also had no effect on protein synthesis in HeLa cells. These results agree well with our finding that 3-methyl-quercetin, a selective

Baciphelacin inhibits translation

80i

Table I. Effect o f baciphelacin on various steps of translation. Additions

[3H]Phe-tRNA binding

P o l y (U) Control + Baciphelacin 10 -4 M + Baciphelacin 10 -5 M -

N-Ac-(3H) Phe-tRNA binding

Poly[3H]Phe synthe~,is

Peptidyl-[3H]puromycin formation

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% control

cpm

% control

cpm

% control

cpm

% control

2484 9555

100

1090 3590

100

407 4428

100

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100

9336

98

3416

95

4220

95

11441

96

9749

102

4372

121

4372

99

12518

105

* Represents a control without polysomes added. Conditions and components of these reactions were as described [11].

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802

L. Carrasco

inhibitor of poliovirus RNA synthesis, had no effect on translation [15]. Finally, terphenyllin, a compound produced by Aspergillus candidus [ 16], and frenolicin, an antibiotic from S t r e p t o m y c e s fradiae [17], were potent inhibitors of protein synthesis in HeLa cells. The latter compounds are structurally unrelated to baciphelacin and it is not yet known if they directly block translation or whether they interfere with another cellular process that indirectly affects protein synthesis. Experiments are underway to distinguish between these possibilities.

pounds, such as mellein, ramulosin, citrinone, sclerotinin-A, oosponol, monocerin, fusamarin, monocerolide, canescin, etc.

Acknowledgments The expert technical assistance of Ms. M.A. Ramos is acknowledged. FISS and CAICYT are acknowledged for financial support.

References Discussion The discovery of new natural compounds is of great interest in many areas of research. Some of these compounds are of therapeutic interest and may advantageously replace existing drugs. Moreover, the continuing search for new compounds and the study of their mode of action provides researchers with powerful tools to dissect complex cellular processes, such as macromolecular synthesis. During the last few years, we have been interested in the discovery of natural agents active against animal viruses [4, 15, 18]. In the course of this search we found that baciphelacin had a potent toxic effect against HeLa cells [4]. The study of its mechanism of action presented in this work led to the conclusion that this antibiotic is an inhibitor of protein synthesis in eukaryotic cells. This compound is most interesting because it blocks translation only in animal cells and has no effect on protein synthesis in plant, yeast or E. coli systems. Although a precedent exists for an antibiotic, tenuazonic acid, which has similar characteristics [2], differences exist between tenuazonic acid as compared to baciphelacin. First of all, tenuazonic acid is a much poorer inhibitor of translation than baciphelacin, which is active in cell-free systems at concentrations 100-fold lower than tenuazonic acid. Second, tenuazonic acid acts on the peptidyl-transferase center located on the ribosome [2], whereas we believe that baciphelacin blocks the charging of tRNA with amino acids similar to ochratoxin-A [19]. The possibility that baciphelacin blocks the initiation step of translation is also open. One of the most significant aspects of this work is the identification of natural compounds related to baciphelacin as potential translation inhibitors. Our finding that ADDC also blocks protein synthesis in HeLa cells, but not in yeast cells suggests that this selectivity can be shared with other c o r n -

1 Vfizquez D. (1979) in: Inhibitors o f Protein Biosynthesis. Springer-Verlag, Heidelberg 2 Carrasco L. & Vfizquez D. (1973) Biochim. Biophys. Acta 319, 209-217 3 Barbacid M. & Vfizquez D. (1974) J. Mol. Biol. 84, 603-623 4 Alarc6n B., Lacal J.C., Fernandez-Soma J.M. & Carrasco L. (1984) Antiviral Res. 4, 231-243 5 Okasaki H., Kishi T., Beppu T. & Arima K. 0975) J. Antibiot. 28, 717-721 6 Carrasco L., Battaner E. & Vazquez D. (1974) Methods Enzymol. 30, 282-289 7 Cabrer B., V~zquez D. & Modolell J. (1972) Proc. Natl. Acad. Sci. USA 69, 733-736 8 Carrasco L. & Vazquez D. 0975) Eur. J. Biochem. 50, 317-323 9 de Haro C., de Herreros A.G. & Ochoa S. (1983) Proc. Natl. Acad. Sci. USA 80, 6843-6847 10 Carrasco L., H a~'vey R , Blanchard Co & Sm__iithAoE~ (1979) in: Modern Trends in Human Leukemia (Neth R., Gallo R.C., Hofschneider P.H. & Mannwereler R., eds.), Springer-Verlag, Heidelberg, pp. 277-281 11 Carrasco L., Fernfindez-Puentes C. & Vfizquez D. (1976) Mcl. Cell. Biochem. 10, 97-122 12 Scott P.M. (1984) in: Mycotoxins: Production, Isolation, Separation and Purification (Betina V., ed.), Elsevier, Amsterdam, pp. 457-461 13 Aldridge D.C., Grove J.F. & Turner W.B. (1966) J. Chem. Soc. (C) 126-129 14 Bird A.E. & Marshall A.C. (1969) d. Chem. Soc. (C) 2418- 2420 15 Castrillo J.L., Vanden Berghe D. & Carrasco L. (1986) Virology 152, 219-227 16 Marchelli R. & Vining L.C. (1975) J. Antibiot. 28, 328-331 17 EUestad G.A., Kunstmann M.P., Whaley H.A. & Patterson E.L. (1968) J. Am. Chem. Soc. 90, 1325-1332 18 Carrasco L. & Vfizquez D. (1983) in" Antibiotics Vol. III. (Hahn F., ed.), Springer-Verlag, Heidelberg, pp. 279-295 19 Creppy E.E., Schlegel M., Roschenthaler R. & Dirheimer G. (1980) Toxicol. Lett. 6, 77-80